The goal of this project is to investigate hindbrain ventricle development. The embryonic brain first forms as a tube, the lumen of which forms the brain ventricles, which are a conserved system of cavities containing cerebrospinal fluid. The ventricular system serves important functions including waste removal and protection. However, we lack a detailed understanding of how the ventricular system forms. Our strategy is to exploit the advantages of the zebrafish model to study this process in vivo by combining time-lapse imaging approaches with gene knockdown methods and the use of mutant and transgenic zebrafish. Aim 1 will characterize the normal temporal and spatial progression of hindbrain ventricle morphogenesis using time-lapse imaging analysis. This study will make use of transgenic embryos expressing GFP in rhombomere (r) 3 and r5, and additionally labeled with a cell membrane marker to allow detailed observation of cellular rearrangements underlying ventricle morphogenesis. Immunohistochemistry on fixed specimens will reveal how cell polarity and epithelial integrity alter during ventricle development. These experiments will confirm the preliminary observation of positional correlation between initial ventricle openings and rhombomere boundaries, determine the order in which ventricle openings form, and establish whether there is differential cell polarity in rhombomere boundary cells. Aim 2 will test the hypothesis that rhombomere boundary formation is required for normal ventricle morphogenesis. Rhombomere boundaries will be disrupted using (1) morpholinos to knockdown the function of EphA4 and/or EphrinB2a and (2) val mutants. Time-lapse analysis will address whether lack of rhombomere boundaries prevents normal ventricle morphogenesis. Immunohistochemistry will address whether loss of boundaries alters cell polarity correlated with disrupted morphogenesis. In the long term, this work will lead to further investigation of how altered cellular behaviors influence ventricle morphogenesis and will increase our understanding of human ventricular defects. Relevance to public health: The ventricular system of the brain is required for normal brain function, and is critical to protect against brain trauma. Several human diseases are characterized by changes to the size of brain ventricles. This proposal is aimed at understanding how the ventricular system develops both in normal and genetically altered embryos. These studies will be of relevance to elucidating the causes and treatments of human diseases associated with ventricular defects. [unreadable] [unreadable]